UNASSIGNED: Older patients who are predisposed to bullous pemphigoid (BP) may exhibit reluctance to undergo skin biopsy due to potential complications.
UNASSIGNED: This study aimed to conduct a comparative evaluation among histology, direct immunofluorescence (DIF), and indirect immunofluorescence (IIF) to determine the optimal diagnostic tool in elderly patients.
UNASSIGNED: A retrospective study was conducted on 841 patients suspected of having BP. All cases were initially classified as BP and non-BP in accordance with the diagnostic criteria. Student\'s t-test and chi-squared test examined differences between the 2 groups. We evaluated the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio detected by the 3 tools. We stratified the analysis by age to compare the performance of the diagnostic tools and examined the risk factors associated with BP using logistic regression.
UNASSIGNED: Overall, histology exhibited the highest sensitivity (89.4%), while DIF demonstrated the highest specificity (67.1%). In the elderly, the IIF test exhibited the highest specificity (57.5%), the highest positive likelihood ratio (2.047), and the lowest negative likelihood ratio (0.226). Among patients taking Dipeptidyl Peptidase-4 (DPP-4) inhibitors, IIF demonstrated the highest positive likelihood ratio (3.194) and the second-lowest negative likelihood ratio (0.235).
UNASSIGNED: In cases that elderly patients suspected of having BP are reluctant to undergo skin biopsy, IIF demonstrates the optimal diagnostic method due to its highest positive likelihood ratio, the lowest negative likelihood ratio among the 3 diagnostic measures. Moreover, IIF is found to be a more effective tool for detecting BP in patients using DPP-4 inhibitors.

BACKGROUND: Indirect immunofluorescence (IIF) plays a crucial role in the diagnosis of pemphigus and bullous pemphigoid (BP) by detecting the presence of circulating autoantibodies in the serum of patients. The standard serum transportation method requires delivery to laboratories at 2-8 °C within a day and storage at -20 to -80 °C. However, this protocol poses logistical challenges.
OBJECTIVE: We conducted a study to assess how temperature variations affect the effectiveness of IIF tests.
METHODS: This case-control study analysed 203 serum specimens: 102 from patients with pemphigus and 101 from patients with BP. Specimens were stored at -80 °C (control), 24 °C, and 40 °C for seven days before analysis to investigate variations in IIF titres compared to the control conditions.
RESULTS: In pemphigus serum, 95% at 24 °C and 76% at 40 °C showed no titre difference compared to controls. Similarly, 89% of BP serum at 24 °C and 82% at 40 °C matched the control titres. While 57 specimens across both groups experienced reduced titres, the decrease was primarily marginal (one-step reduction in 54 cases, two-step in 3), with no transition from positive to negative results.
CONCLUSIONS: Storing serum at 24-40 °C for up to seven days before testing slightly influences IIF outcomes for pemphigus and BP. These findings could prompt a significant revision in the existing strict transport guidelines, ensuring efficient use of resources without sacrificing the accuracy of diagnostic tests.

Smooth muscle antibodies (SMA) with anti-microfilament actin (MF-SMA) specificity are regarded as highly specific markers of type 1 autoimmune hepatitis (AIH-1) but their recognition relying on immunofluorescence of vessel, glomeruli, and tubules (SMA-VGT pattern) in rodent kidney tissue, is restricted by operator-dependent interpretation. A gold standard method for their identification is not available. We assessed and compared the diagnostic accuracy for AIH-1 of an embryonal aorta vascular smooth muscle (VSM) cell line-based assay with those of the rodent tissue-based assay for the detection of MF-SMA pattern in AIH-1 patients and controls. Sera from 138 AIH-1 patients and 295 controls (105 primary biliary cholangitis, 40 primary sclerosing cholangitis, 50 chronic viral hepatitis, 20 alcohol-related liver disease, 40 steatotic liver disease, and 40 healthy controls) were assayed for MF-SMA and SMA-VGT using VSM-based and rodent tissue-based assays, respectively. MF-SMA and SMA-VGT were found in 96 (70%) and 87 (63%) AIH-1 patients, and 2 controls (P < 0.0001). Compared with SMA-VGT, MF-SMA showed similar specificity (99%), higher sensitivity (70% vs 63%, P = ns) and likelihood ratio for a positive test (70 vs 65). Nine (7%) AIH-1 patients were MF-SMA positive despite being SMA-VGT negative. Overall agreement between SMA-VGT and MF-SMA was 87% (kappa coefficient 0.870, [0.789-0.952]). MF-SMA were associated with higher serum γ-globulin [26 (12-55) vs 20 g/l (13-34), P < 0.005] and immunoglobulin G (IgG) levels [3155 (1296-7344) vs 2050 mg/dl (1377-3357), P < 0.002]. The easily recognizable IFL MF-SMA pattern on VSM cells strongly correlated with SMA-VGT and has an equally high specificity for AIH-1. Confirmation of these results in other laboratories would support the clinical application of the VSM cell-based assay for reliable detection of AIH-specific SMA.

Background and Objectives: Age-related macular degeneration (AMD) is one of the leading causes of central vision loss among elderly patients, and its dry form accounts for the majority of cases. Although several causes and mechanisms for the development and progression of AMD have previously been identified, the pathogenesis of this complex disease is still not entirely understood. As inflammation and immune system involvement are strongly suggested to play a central role in promoting the degenerative process and stimulating the onset of complications, we aimed to analyze the frequency of serum anti-retinal (ARAs) and anti-endothelial cell antibodies (AECAs) in patients with dry AMD and to determine their relationship with the clinical features of the disease, notably the area of geographic atrophy (GA). Materials and Methods: This study included 41 patients with advanced-stage dry AMD and 50 healthy controls without AMD, matched for gender and age. ARAs were detected by indirect immunofluorescence using monkey retina as an antigen substrate, and the presence of AECAs was determined using cultivated human umbilical vein endothelial cells and primate skeletal muscle. Results: ARAs were detected in 36 (87.8%) AMD patients (titers ranged from 1:20 to 1:320) and in 16 (39.0%) (titers ranged from 1:10 to 1:40) controls (p = 0.0000). Twenty of the forty-one patients (48.8%) were positive for AECAs, while in the control group, AECAs were present only in five sera (10.0%). The titers of AECAs in AMD patients ranged from 1:100 to 1:1000, and in the control group, the AECA titers were 1:100 (p = 0.0001). There were no significant correlations between the presence of AECAs and disease activity. Conclusions: This study demonstrates a higher prevalence of circulating AECAs in patients with dry AMD; however, no correlation was found between the serum levels of these autoantibodies and the area of GA.

QseC is a membrane sensor kinase that enables bacteria to perceive autoinducers -3, adrenaline, and norepinephrine to initiate downstream gene transcription. In this study, we found that the QseC protein of Glaesserella parasuis can serve as an effective antigen to activate the host\'s immune response. Therefore, we investigated the immunogenicity and host protective effect of this protein. ELISA and indirect immunofluorescence results showed that QseC protein can induce high titer levels of humoral immunity in mice and regularly generate specific serum antibodies. We used MTS reagents to detect lymphocyte proliferation levels and found that QseC protein can cause splenic lymphocyte proliferation with memory and specificity. Further immunological analysis of the spleen cell supernatant revealed significant upregulation of levels of IL-1β, IL-4 and IFN-γ in the QseC + adjuvant group. In the mouse challenge experiment, it was found that QseC + adjuvant can provide effective protection. The results of this study demonstrate that QseC protein provides effective protection in a mouse model and has the potential to serve as a candidate antigen for a novel subunit vaccine for further research.

Cell division is a conserved process among eukaryotes. It is designed to segregate chromosomes into future daughter cells and involves a complex rearrangement of the cytoskeleton, including microtubules and actin filaments. An additional level of complexity is present in asymmetric dividing stem cells because cytoskeleton elements are also regulated by polarity cues. The neural stem cell system of the fruit fly represents a simple model to dissect the mechanisms that control cytoskeleton reorganization during asymmetric division. In this chapter, we propose to describe protocols that allow accurate analysis of microtubule reorganization during cell division in this model.

The combination of patterns is a frequent and challenging situation in the daily laboratory routine of autoantibodies testing using HEp-2 cells indirect immunofluorescence assay (HEp-2-IFA). Recently, the Brazilian Consensus on Autoantibodies (BCA) named these combinations as complex patterns (CPs) and organized them into 3 subtypes: multiple, mixed, and composite. This study aimed to describe the most frequent combinations of HEp-2-IIF patterns according to this new nomenclature.
Routine HEp-2-IFA results reported in January and June 2017 were reviewed using the new BCA classification. Visual pattern recognition was performed by experts on HEp-2-IFA readings, using the International Consensus on Antinuclear Antibodies (ANA) Patterns (ICAP) and BCA recommendations.
54,990 serum samples from different patients were tested for ANA-HEp-2, and 11,478 (20.9%) were positive at a titer ≥ 1/80. Among these positive samples, 1,111 (9.7%) displayed CPs, divided into 95 different combinations. A higher proportion of CPs was observed in the pediatric age group. Multiple, mixed, and composite patterns were present in 85.3, 5.4, and 9.5% of the samples, respectively. In the multiple/mixed pattern group (n=1,005), double, triple, and quadruple combinations (ICAP/BCA codes) were observed in 97.7%, 2.2%, and 0.1%, respectively. The double nuclear pattern was the most prevalent combination observed (67.6%). The most common CPs registered were AC-4 (nuclear fine speckled) + AC-6,7 (nuclear discrete dots) (n=264); AC-2 (nuclear dense fine speckled) + AC-6,7 (n=201); AC-4+AC-8,9,10 (nucleolar) (n=129); and AC-3 (centromere)+AC-4 (n=124). All of these combinations were in the multiple subgroup.
Almost 10% of positive results in the HEp-2 procedure displayed CPs. Among the 3 subtypes of CPs proposed, the multiple pattern was the most prevalent, especially in the pediatric population. The AC-4, AC-2, and AC-6,7 were the most prevalent single patterns observed in the combinations described in this study. There was a significant association between age and the prevalence of most combined patterns. The AC-4+AC-6,7 combination was the most prevalent complex pattern detected regardless of the age group. The AC-2+AC-6,7 was more prevalent in younger individuals. The concepts involved in the CPs definition should add value to the reading and interpretation of the HEp-2-IIF assay.

Rare antinuclear antibody (ANA) pattern recognition has been a widely applied technology for routine ANA screening in clinical laboratories. In recent years, the application of deep learning methods in recognizing ANA patterns has witnessed remarkable advancements. However, the majority of studies in this field have primarily focused on the classification of the most common ANA patterns, while another subset has concentrated on the detection of mitotic metaphase cells. To date, no prior research has been specifically dedicated to the identification of rare ANA patterns. In the present paper, we introduce a novel attention-based enhancement framework, which was designed for the recognition of rare ANA patterns in ANA-indirect immunofluorescence images. More specifically, we selected the algorithm with the best performance as our target detection network by conducting comparative experiments. We then further developed and enhanced the chosen algorithm through a series of optimizations. Then, attention mechanism was introduced to facilitate neural networks in expediting the learning process, extracting more essential and distinctive features for the target features that belong to the specific patterns. The proposed approach has helped to obtained high precision rate of 86.40%, 82.75% recall, 84.24% F1 score and 84.64% mean average precision for a 9-category rare ANA pattern detection task on our dataset. Finally, we evaluated the potential of the model as medical technologist assistant and observed that the technologist\'s performance improved after referring to the results of the model prediction. These promising results highlighted its potential as an efficient and reliable tool to assist medical technologists in their clinical practice.

BACKGROUND: Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Detection of antinuclear antibodies (ANAs) aids in the diagnosis of SLE. The indirect immunofluorescence (IIF) assay is often used a routine screening test for the detection of ANA. The pathogenic role and significance of various patterns produced in IIF is yet to be explored.
OBJECTIVE: This study aimed to detect ANA patterns generated by IIF and correlate these patterns with specific antibodies detected by line immunoassay. We also investigated the significance of each ANA pattern and its association with specific serological SLE markers, such as complement molecules, anti-dsDNA, antiphospholipid antibody, and C-reactive protein (CRP), along with associations with direct Coombs test (DCT).
METHODS: We conducted a retrospective study that included 204 patients newly diagnosed with SLE according to the European Alliance of Associations for Rheumatology/American College of Rheumatology (EULAR/ACR) criteria. The detection and pattern determination of ANA was performed by IIF using HEp-20-10. Furthermore, line immunoassay was performed, and the antibody profile of each sample was obtained. Other immunodiagnostic markers were analyzed, including C3, C4, anti-dsDNA, antiphospholipid antibodies (anti-cardiolipin antibodies, anti-beta-2-glycoprotein I, and lupus anticoagulant), CRP, and DCT.
RESULTS: Of the 204 samples, the most frequent ANA pattern observed was nucleus speckled (52.9%), followed by nucleus homogenous (27.5%), mixed (13.7%), and cytoplasm speckled (5.9%). The nucleus homogenous pattern showed the most pathogenic immune profile due to its close association with markers of disease activity, namely, high anti-dsDNA titer, low C3 level, and DCT positivity.  Conclusion: This study showed that the most common pattern associated with SLE is nucleus speckled, followed by the nucleus homogenous pattern. Based on associations with specific serological markers, the nucleus homogenous pattern may be linked to a high disease activity in SLE.

BACKGROUND: A major challenge of the HEp-2 cell-based indirect immunofluorescence (IIF) assays is the correct identification of the individual anti-nuclear antibodies (ANAs) if more than one is present in a sample. We created artificial mixes by pooling two different samples with a single autoantibody in different titers. Comparison of the expected and observed patterns and titers clarifies the interference between the two tested ANAs.
METHODS: Serum samples with a single homogeneous or speckled ANA pattern were serially diluted and mixed in 16 combinations, providing end-point titers of 1:5,120 to 1:80 for both patterns. These mixes were tested by a HEp-2 IIF assay and were evaluated by conventional evaluation, the EUROPattern (EPa) system and on-screen analysis.
RESULTS: Homogeneous pattern can alter the identification of the speckled pattern much more than vice versa, but both has an interfering effect on the other. The effect of the interfering on the tested pattern was higher if the titer of the former one was higher. The pattern recognition efficacy of conventional and the on-screen evaluation was similar and superior compared to the EPa analysis.
CONCLUSIONS: The application of artificial mixed samples can help the evaluation of the efficacy of manual and computer-aided ANA HEp-2 pattern recognition.